Title: Capturing the genomic variation present in Cryptosporidium and cryptosporidiosis PROJECT SUMMARY Parasites of the genus Cryptosporidium cause 748,000 cases of cryptosporidiosis annually in the U.S. and are the second-leading cause of diarrheal disease in infants worldwide. Globally in 2016, among children younger than 5, acute cryptosporidiosis caused 48,000 deaths and 4.2 million disability-adjusted life-years (DALYs) lost with 7.8 million more DALYs attributable to growth faltering. Cryptosporidium species are eukaryotic apicomplexan parasites classified as bio-defense category B pathogens. Waterborne outbreaks of Cryptosporidium have caused up to 400,000 illnesses in a single incident in the U.S., and are a constant threat to public health worldwide. The CDC estimates a >3-fold increase in cases of cryptosporidiosis in the U.S. since 2004. To address this urgent public health problem, we propose to develop and use new genomic resources to better understand Cryptosporidium species that infect humans by characterizing the distribution of DNA variants in local and global contexts. We will sequence ~1500 samples from a vast set of >53,000 curated samples to generate broadly-applicable population genetic data and genomic resources for species that have thus far been refractory to extensive sequence characterization. The proposed studies and resources will take the Cryptosporidium research community to the next level, effectively changing the types of questions that can be asked by providing a new context to generate hypotheses and new tools that can be re-used to further our knowledge of these pathogens. Specifically, AIM 1 will develop refined hybrid capture bait sets for powerful new assays to characterize Cryptosporidium. We will develop two bait sets for capture enriched sequencing (CES), one for whole genome characterization (focusing on species that most frequently infect humans) and one for high-throughput characterization of all Cryptosporidium species that commonly infect humans. CES is a game-changer for Cryptosporidium research, facilitating multi-locus and whole genome characterization from fecal DNA or environmental samples where Cryptosporidium DNA is low in abundance. AIM 2 will generate and improve genome assemblies and annotation for C. hominis, C. tyzzeri, C. meleagridis, and C. cuniculus. These assemblies complete the reference sequences needed for species that commonly infect humans (or are a model) and will serve as a reference for the data that will be generated in Aim 3 and as a resource for the biomedical research community. AIM 3 will characterize the genetic diversity of C. parvum and C. hominis through surveys of the distribution of genomic diversity in specific contexts. We focus on these two species because they cause >90% cryptosporidiosis cases. We will conduct high-throughput CES on 1152 samples, identifying 384 of those plus 384 additional samples for full genome CES. We will use the resulting data to test hypotheses on: Cryptosporidium population structure, mixed infections, recombination, and the evolution of anthroponotic strains. This project will generate game-changing new tools for the Cryptosporidium research community and provide new insight into the biology of parasites that sicken thousands of children daily.